It has become increasingly difficult to find an area of biology and medicine in which membrane lipids do not play important signaling and regulatory roles. Phosphorylated derivatives of phosphatidylinositol, collectively known as phosphoinositides (PtdInsPs), play key roles in diverse cellular processes. The importance of PtdInsP-mediated cell regulation has been demonstrated by numerous human diseases linked to defects in PtdInsP signaling, including cancer, diabetes, and inflammatory diseases. Consequently, the PtdInsP-mediated cell signaling pathways are major targets for drug development. However, it is still poorly understood how PtdInsPs specifically regulate such diverse cellular processes and the lack of this fundamental understanding greatly undermines the effort to develop specific and potent therapeutic agents targeted against PtdInsP signaling pathways. Based on our recent studies, we hypothesize that local PtdInsP concentrations act as differential activation thresholds, triggering diverse downstream cellular processes. The primary objective of this proposed research is to investigate the complex mechanisms underlying diverse PtdInsP-mediated cell regulation on the basis of this testable hypothesis and using our newly developed lipid sensor technology that allows in situ quantification of PtdInsP in live cels. Specificaly, we propose to (1) develop new fluorescence probes and imaging techniques for quantifying PtdInsPs in mammalian cells, individually and in combination, (2) determine the mechanisms of differential cell regulation by two key signaling lipids, phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol-3,4- bisphosphate, and (3) determine of the role of another important lipid, phosphatidylserine, in phosphatidylinositol 3,4,5-trisphosphate signaling. PUBLIC HEALTH RELEVANCE: Phosphoinositides are lipid molecules that play important signaling and regulatory roles in diverse cellular processes, including cell proliferation, apoptosis, metabolism and migration. Perturbations in the phosphoinositide-mediated cell regulation contribute to the pathogenesis of human diseases including inflammation, cancer, diabetes and metabolic diseases. Since phosphoinositides are dynamic molecules that are produced, degraded, and transported in a tightly controlled manner, determination of their concentration and movement in a temporally and spatially resolved manner is a key step toward the understanding of a growing myriad of phosphoinositide-mediated cellular processes and the development of new strategies to diagnose, treat, and prevent human diseases caused by dysfunctional phosphoinositide-associated processes. We have developed an innovative new fluorescence imaging technology that allows accurate and sensitive lipid quantification in live cells and will apply this technology to the investigation of the complex mechanisms of PtdInsP- mediated cell regulation.